RESUMEN
The dynamic interplay of post-translational modifications (PTMs) in chromatin provides a communication system for the regulation of gene expression. An increasing number of studies have highlighted the role that such crosstalk between PTMs plays in chromatin recognition. In this study, (bio)chemical and structural approaches were applied to specifically probe the impact of acetylation of Lys18 in the histone H3 tail peptide on peptide recognition by the protein methyltransferase coactivator-associated arginine methyltransferase 1 (CARM1). Peptidomimetics that recapitulate the transition state of protein arginine N-methyltransferases, were designed based on the H3 peptide wherein the target Arg17 was flanked by either a free or an acetylated lysine. Structural studies with these peptidomimetics and the catalytic domain of CARM1 provide new insights into the binding of the H3 peptide within the enzyme active site. While the co-crystal structures reveal that lysine acetylation results in minor conformational differences for both CARM1 and the H3 peptide, acetylation of Lys18 does lead to additional interactions (Van der Waals and hydrogen bonding) and likely reduces the cost of desolvation upon binding, resulting in increased affinity. Informed by these findings a series of smaller peptidomimetics were also prepared and found to maintain potent and selective CARM1 inhibition. These findings provide new insights both into the mechanism of crosstalk between arginine methylation and lysine acetylation as well as towards the development of peptidomimetic CARM1 inhibitors.
Asunto(s)
Diseño de Fármacos , Inhibidores Enzimáticos/farmacología , Lisina/antagonistas & inhibidores , Peptidomiméticos/farmacología , Proteína-Arginina N-Metiltransferasas/antagonistas & inhibidores , Acetilación , Animales , Cristalografía por Rayos X , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Lisina/metabolismo , Ratones , Modelos Moleculares , Peptidomiméticos/síntesis química , Peptidomiméticos/química , Conformación Proteica , Proteína-Arginina N-Metiltransferasas/metabolismo , Especificidad por SustratoRESUMEN
The emergence of the C797S mutation in EGFR is a frequent mechanism of resistance to osimertinib in the treatment of non-small cell lung cancer (NSCLC). In the present work, we report the design, synthesis and biochemical characterization of UPR1444 (compound 11), a new sulfonyl fluoride derivative which potently and irreversibly inhibits EGFRL858R/T790M/C797S through the formation of a sulfonamide bond with the catalytic residue Lys745. Enzymatic assays show that compound 11 displayed an inhibitory activity on EGFRWT comparable to that of osimertinib, and it resulted more selective than the sulfonyl fluoride probe XO44, recently reported to inhibit a significant part of the kinome. Neither compound 11 nor XO44 inhibited EGFRdel19/T790M/C797S triple mutant. When tested in Ba/F3 cells expressing EGFRL858R/T790M/C797S, compound 11 resulted significantly more potent than osimertinib at inhibiting both EGFR autophosphorylation and proliferation, even if the inhibition of EGFR autophosphorylation by compound 11 in Ba/F3 cells was not long lasting.
Asunto(s)
Lisina/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Ácidos Sulfínicos/farmacología , Animales , Biocatálisis , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/genética , Receptores ErbB/metabolismo , Humanos , Lisina/metabolismo , Ratones , Modelos Moleculares , Estructura Molecular , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Relación Estructura-Actividad , Ácidos Sulfínicos/síntesis química , Ácidos Sulfínicos/químicaRESUMEN
Human sirtuin 5 (SIRT5) plays pivotal roles in metabolic pathways and other biological processes, and is involved in several human diseases including cancer. Development of new potent and selective SIRT5 inhibitors is currently desirable to provide potential therapeutics for related diseases. Herein, we report a series of new 3-thioureidopropanoic acid derivatives, which were designed to mimic the binding features of SIRT5 glutaryl-lysine substrates. Structure-activity relationship studies revealed several compounds with low micromolar inhibitory activities to SIRT5. Computational and biochemical studies indicated that these compounds exhibited competitive SIRT5 inhibition with respect to the glutaryl-lysine substrate rather than nicotinamide adenine dinucleotide cofactor. Moreover, they showed high selectivity for SIRT5 over SIRT1-3 and 6 and could stabilize SIRT5 proteins as revealed by thermal shift analyses. This work provides an effective substrate-mimicking strategy for future inhibitor design, and offers new inhibitors to investigate their therapeutic potentials in SIRT5-associated disease models.
Asunto(s)
Descubrimiento de Drogas , Inhibidores Enzimáticos/farmacología , Lisina/antagonistas & inhibidores , Propionatos/farmacología , Sirtuinas/antagonistas & inhibidores , Tiourea/farmacología , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Humanos , Lisina/metabolismo , Estructura Molecular , Propionatos/síntesis química , Propionatos/química , Sirtuinas/metabolismo , Relación Estructura-Actividad , Especificidad por Sustrato , Tiourea/síntesis química , Tiourea/químicaRESUMEN
Advanced glycation end products (AGE), compounds formed in meat at the advanced stage of Maillard reaction, are easily exposed to thermal processing. Improving cooking condition and adding antioxidants are 2 common ways for AGE reduction. The present work compared the inhibition of grape seed extract (GSE) on levels of free and protein-bound Nε-carboxymethyllysine (CML) and Nε-carboxyethyllysine (CEL) in chicken breast under deep-frying and air-frying conditions. Efficiency of 5 concentrations of GSE (0.0, 0.2, 0.5, 0.8, and 1.0 g/kg) in retarding oxidation, glyoxal (GO), methylglyoxal (MGO), lysine (Lys), Maillard reaction degree (A294, A420), and Shiff's base were tested. Results showed that 0.5 g/kg GSE before heating significantly (P < 0.05) reduced AGE in fried breast chicken, whereas excessive supplementation of GSE (0.8 and 1 g/kg) was reverse. Air frying was found significantly (P < 0.05) better than deep frying to reduce the precursor substances (GO, MGO, and Lys) of AGE. In conclusion, GSE-derived polyphenols exhibited different inhibitory effects on oxidation and glycosylation at different concentrations. We found that 0.5 g/kg of GSE combined with air frying was the best recommendation for inhibiting CML and CEL.
Asunto(s)
Culinaria/métodos , Extracto de Semillas de Uva/farmacología , Lisina/análogos & derivados , Carne/normas , Animales , Pollos , Productos Finales de Glicación Avanzada/análisis , Productos Finales de Glicación Avanzada/metabolismo , Glicosilación , Lisina/antagonistas & inhibidores , Lisina/metabolismo , Reacción de Maillard , Fibras Musculares Esqueléticas/citología , Oxidación-Reducción , Compuestos de Sulfhidrilo/análisis , Sustancias Reactivas al Ácido Tiobarbitúrico/análisisRESUMEN
In eukaryotes, the polyamine pathway generates spermidine that activates the hypusination of the translation factor eukaryotic initiation factor 5A (eIF5A). Hypusinated-eIF5A modulates translation, elongation, termination and mitochondrial function. Evidence in model organisms like drosophila suggests that targeting polyamines synthesis might be of interest against ischemia. However, the potential of targeting eIF5A hypusination in stroke, the major therapeutic challenge specific to ischemia, is currently unknown. Using in vitro models of ischemic-related stress, we documented that GC7, a specific inhibitor of a key enzyme in the eIF5A activation pathway, affords neuronal protection. We identified the preservation of mitochondrial function and thereby the prevention of toxic ROS generation as major processes of GC7 protection. To represent a thoughtful opportunity of clinical translation, we explored whether GC7 administration reduces the infarct volume and functional deficits in an in vivo transient focal cerebral ischemia (tFCI) model in mice. A single GC7 pre- or post-treatment significantly reduces the infarct volume post-stroke. Moreover, GC7-post-treatment significantly improves mouse performance in the rotarod and Morris water-maze, highlighting beneficial effects on motor and cognitive post-stroke deficits. Our results identify the targeting of the polyamine-eIF5A-hypusine axis as a new therapeutic opportunity and new paradigm of research in stroke and ischemic diseases.
Asunto(s)
Guanina/análogos & derivados , Lisina/análogos & derivados , Mitocondrias/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/antagonistas & inhibidores , Factores de Iniciación de Péptidos/metabolismo , Proteínas de Unión al ARN/metabolismo , Accidente Cerebrovascular/terapia , Animales , Conducta Animal/efectos de los fármacos , Cognición/efectos de los fármacos , Guanina/administración & dosificación , Guanina/farmacología , Guanina/uso terapéutico , Inyecciones Intraperitoneales , Ataque Isquémico Transitorio/tratamiento farmacológico , Ataque Isquémico Transitorio/prevención & control , Lisina/antagonistas & inhibidores , Masculino , Ratones , Ratones Endogámicos C57BL , Mitocondrias/ultraestructura , Modelos Animales , Neuroprotección/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Factores de Iniciación de Péptidos/efectos de los fármacos , Poliaminas/metabolismo , Proteínas de Unión al ARN/efectos de los fármacos , Especies Reactivas de Oxígeno/toxicidad , Accidente Cerebrovascular/metabolismo , Factor 5A Eucariótico de Iniciación de TraducciónRESUMEN
No disponible
Asunto(s)
Humanos , Antifibrinolíticos/administración & dosificación , Transfusión Sanguínea/tendencias , Procedimientos Ortopédicos/métodos , Ácido Tranexámico/administración & dosificación , Antifibrinolíticos/efectos adversos , Artroplastia de Reemplazo de Cadera/métodos , Artroplastia de Reemplazo de Rodilla/métodos , Pérdida de Sangre Quirúrgica/prevención & control , Fibrinólisis , Fracturas de Cadera/sangre , Fracturas de Cadera/cirugía , Lisina/antagonistas & inhibidores , Metaanálisis como Asunto , Procedimientos Ortopédicos/efectos adversos , Hemorragia Posoperatoria/sangre , Hemorragia Posoperatoria/prevención & control , Columna Vertebral/cirugía , Ácido Tranexámico/efectos adversosAsunto(s)
Antifibrinolíticos/administración & dosificación , Transfusión Sanguínea/tendencias , Procedimientos Ortopédicos/métodos , Ácido Tranexámico/administración & dosificación , Antifibrinolíticos/efectos adversos , Artroplastia de Reemplazo de Cadera/métodos , Artroplastia de Reemplazo de Rodilla/métodos , Pérdida de Sangre Quirúrgica/prevención & control , Fibrinólisis , Fracturas de Cadera/sangre , Fracturas de Cadera/cirugía , Humanos , Lisina/antagonistas & inhibidores , Metaanálisis como Asunto , Procedimientos Ortopédicos/efectos adversos , Hemorragia Posoperatoria/sangre , Hemorragia Posoperatoria/prevención & control , Columna Vertebral/cirugía , Ácido Tranexámico/efectos adversosRESUMEN
Advanced glycation endproducts (AGEs) can be found in protein- and sugar-rich food products processed at high temperatures, which make up a vast amount of the Western diet. The effect of AGE-rich food products on human health is not yet clear and controversy still exists due to possible contamination of samples with endotoxin and the use of endogenous formed AGEs. AGEs occur in food products, both as protein-bound and individual molecules. Which form exactly induces a pro-inflammatory effect is also unknown. In this study, we exposed human macrophage-like cells to dietary AGEs, both in a protein matrix and individual AGEs. It was ensured that all samples did not contain endotoxin concentrations > 0.06 EU/mL. The dietary AGEs induced TNF-alpha secretion of human macrophage-like cells. This effect was decreased by the addition of N(ε)-carboxymethyllysine (CML)-antibodies or a receptor for advanced glycation endproducts (RAGE) antagonist. None of the individual AGEs induce any TNF-alpha, indicating that AGEs should be bound to proteins to exert an inflammatory reaction. These findings show that dietary AGEs directly stimulate the inflammatory response of human innate immune cells and help us define the risk of regular consumption of AGE-rich food products on human health.
Asunto(s)
Dieta Occidental/efectos adversos , Productos Finales de Glicación Avanzada/efectos adversos , Inflamación/inducido químicamente , Macrófagos/efectos de los fármacos , Macrófagos/fisiología , Receptor para Productos Finales de Glicación Avanzada/antagonistas & inhibidores , Anticuerpos/farmacología , Caseínas/química , Caseínas/farmacología , Endotoxinas/análisis , Contaminación de Alimentos/análisis , Productos Finales de Glicación Avanzada/farmacología , Glicosilación , Calor , Humanos , Lisina/análogos & derivados , Lisina/antagonistas & inhibidores , Lisina/inmunología , Macrófagos/inmunología , Reacción de Maillard , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Nε-(carboxymethyl) lysine (CML) plays causal roles in diabetic complications. In the present study, we investigated whether CML-induced HIF-1α accumulation and epithelial-mesenchymal transition (EMT) in HK-2 renal proximal tubular epithelial cells. Treatment with CML-BSA increased reactive oxygen species (ROS) production reduced the mitochondrial membrane potential and induced mitochondrial fragmentation. Pre-treatment of cells with antioxidant, α-lipoic acid, normalised the ROS production and restored the mitochondrial membrane potential. These changes were accompanied with morphological changes of epithelial mesenchymal transition. CML-BSA increased the protein level of hypoxia-inducible factor-1α (HIF-1α), and the EMT-associated transcription factor, TWIST. These effects were reversed by α-lipoic acid. CML-BSA increased the protein levels of mesenchymal-specific markers, including vimentin, α-smooth muscle actin, which were alleviated by pre-treatment with α-lipoic acid. Our data suggest that CML-BSA induces EMT through a ROS/HIF-1α/TWIST-dependent mechanism, and that α-lipoic acid may alleviate the CML-induced EMT in renal tubular cells.
Asunto(s)
Transición Epitelial-Mesenquimal/efectos de los fármacos , Túbulos Renales Proximales/efectos de los fármacos , Lisina/análogos & derivados , Ácido Tióctico/farmacología , Células Cultivadas , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Túbulos Renales Proximales/metabolismo , Lisina/antagonistas & inhibidores , Lisina/farmacología , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Lysine-specific demethylase 1 (LSD1) is a flavin-dependent enzyme that removes methyl groups from mono- or dimethylated lysine residues at the fourth position of histone H3. We have previously reported several histone H3 peptides containing an LSD1 inactivator motif at Lys-4. In this study, histone H3 peptides having a trans-2-phenylcyclopropylamine (PCPA), a 2,5-dihydro-1H-pyrrole, and a 1,2,3,6-tetrahydropyridine moiety at Lys-4 were prepared along with related compounds possessing a shorter side chain at the fourth position. Enzymatic assays showed that PCPA peptides containing a longer side chain, which can react with FAD in the active site, are potent LSD1-selective inhibitors.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Histona Demetilasas/antagonistas & inhibidores , Histonas/farmacología , Lisina/antagonistas & inhibidores , Péptidos/farmacología , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Histona Demetilasas/metabolismo , Histonas/química , Humanos , Lisina/metabolismo , Estructura Molecular , Péptidos/química , Relación Estructura-ActividadRESUMEN
Advanced Glycation End-products (AGEs) have been associated to diabetes, neurodegenerative and cardiovascular diseases. Mitigating the formation of AGEs is a strategy to avoid detrimental physiopathological effects of age-related chronic diseases. An olive leaf extract (OLE), obtained under acidic conditions, and two fractions, obtained by solid-phase extraction, were characterized by LC-MS/MS. Antiglycative capacity of OLE and fractions were investigated in different in vitro models. The OLE significantly inhibited the formation of Amadori products at the early stage as well as the formation of fluorescent AGEs at the advanced stage of the glycation. Carboxymethyllysine was significantly inhibited by the OLE but it showed weaker activity against argpyrimidine and carboxyethyllysine. The antiglycative activity of each OLE fraction independently did not explain the activity reached in the whole extract, being necessary the compounds present in both fractions. OLE and its fractions were highly effective for trapping reactive dicarbonyl compounds (glyoxal, methylglyoxal, 3-deoxyglucosone and 3-deoxygalactosone). Different adducts resulting from the conjugation of methylglyoxal and hydroxytyrosol in OLE were identified. Results pointed out that OLE exert a broad-spectrum in vitro antiglycative activity.
Asunto(s)
Olea/química , Extractos Vegetales/farmacología , Hojas de la Planta/química , Cromatografía Liquida , Desoxiglucosa/análogos & derivados , Desoxiglucosa/metabolismo , Fructosamina/antagonistas & inhibidores , Fructosamina/metabolismo , Galactosa/análogos & derivados , Galactosa/metabolismo , Productos Finales de Glicación Avanzada/antagonistas & inhibidores , Productos Finales de Glicación Avanzada/metabolismo , Glicosilación , Glioxal/metabolismo , Lisina/análogos & derivados , Lisina/antagonistas & inhibidores , Lisina/metabolismo , Ornitina/análogos & derivados , Ornitina/antagonistas & inhibidores , Ornitina/metabolismo , Fenoles/farmacología , Alcohol Feniletílico/análogos & derivados , Alcohol Feniletílico/metabolismo , Pirimidinas/antagonistas & inhibidores , Pirimidinas/metabolismo , Piruvaldehído/metabolismo , Espectrometría de Masas en TándemRESUMEN
Human lysine demethylase (KDM) enzymes (KDM1-7) constitute an emerging class of therapeutic targets, with activities that support growth and development of metastatic disease. By interacting with and co-activating the androgen receptor, the KDM4 subfamily (KDM4A-E) promotes aggressive phenotypes of prostate cancer (PCa). Knockdown of KDM4 expression or inhibition of KDM4 enzyme activity reduces the proliferation of PCa cell lines and highlights inhibition of lysine demethylation as a possible therapeutic method for PCa treatment. To address this possibility, we screened the ChemBioNet small molecule library for inhibitors of the human KDM4E isoform and identified several compounds with IC50 values in the low micromolar range. Two hits, validated as active by an orthogonal enzyme-linked immunosorbent assay, displayed moderate selectivity toward the KDM4 subfamily and exhibited antiproliferative effects in cellular models of PCa. These compounds were further characterized by their ability to maintain the transcriptionally silent histone H3 tri-methyl K9 epigenetic mark at subcytotoxic concentrations. Taken together, these efforts identify and validate a hydroxyquinoline scaffold and a novel benzimidazole pyrazolone scaffold as tractable for entry into hit-to-lead chemical optimization campaigns.
Asunto(s)
Bencimidazoles/farmacología , Proliferación Celular/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Histona Demetilasas con Dominio de Jumonji/antagonistas & inhibidores , Lisina/antagonistas & inhibidores , Neoplasias de la Próstata/tratamiento farmacológico , Pirazolonas/farmacología , Línea Celular Tumoral , Desmetilación/efectos de los fármacos , Histona Demetilasas/metabolismo , Histonas/metabolismo , Humanos , Hidroxiquinolinas/farmacología , Masculino , Células PC-3 , Neoplasias de la Próstata/metabolismo , Receptores Androgénicos/metabolismo , Transcripción Genética/efectos de los fármacosRESUMEN
Human band 3 is a 98-kDa transmembrane (TM) protein comprising 14 TM segments. Papain cleavages band 3 into 38- and 60-kDa fragments. Under vigorous conditions, the cleavage of the loop region between the TM 7 of gate domain and the TM 8 of core domain in the 38-kDa fragment produces 7- and 31-kDa fragments. Conformational changes of the TM 5 segment containing Lys-539 by cleavage of the 38-kDa fragment remain unclear. Pressure-induced haemolysis of erythrocytes was suppressed by binding of 4, 4'-diisothiocyanostilbene-2, 2'-disulfonate (DIDS) to Lys-539. Such effect of DIDS was not observed upon cleavage of the 38-kDa fragment, because of inhibition of DIDS binding to Lys-539. Using fluorescence of DIDS labelled to Lys-539, conformational changes of band 3 were examined. Fluorescence spectra demonstrated that the molecular motion of DIDS is more restricted upon digestion of the 38-kDa fragment. Interestingly, the quenching of DIDS fluorescence showed that Hg2+ is less accessible to DIDS upon digestion of the 38-kDa fragment. Taken together, we propose that the conformational changes of the TM 5 segment characterized by the sequestration and restricted motion of Lys-539 are induced by the cleavage of the loop region between the TM 7 and the TM 8.
Asunto(s)
Ácido 4,4'-Diisotiocianostilbeno-2,2'-Disulfónico/antagonistas & inhibidores , Proteína 1 de Intercambio de Anión de Eritrocito/antagonistas & inhibidores , Lisina/antagonistas & inhibidores , Papaína/farmacología , Fragmentos de Péptidos/efectos de los fármacos , Ácido 4,4'-Diisotiocianostilbeno-2,2'-Disulfónico/química , Proteína 1 de Intercambio de Anión de Eritrocito/química , Sitios de Unión/efectos de los fármacos , Humanos , Lisina/químicaRESUMEN
The antiglycative activity of hydroxytyrosol (HT) and olive leaf extract (OLE) was investigated in wheat-flour biscuits. Quercetin (QE) and gallic acid (GA) were used as reference of antiglycative activity of phenolic compounds. HT, OLE, QE and GA were added in the range of 0.25-0.75% (w/w). Samples were compared against a control recipe baked at 180°C/20min. HT biscuit was able to inhibit efficiently the formation of hydroxymethylfurfural (HMF) and 3-deoxyglucosone (3-DG), as well as reduced the formation of overall free fluorescent AGEs and pentosidine. The inhibition of the 3-DG and HMF formation was directly and significantly correlated under controlled baking conditions. However, samples formulated with OLE exerted similar antiglycative capacity against pentosidine and Nε-carboxyethyl-lysine, although the amount of HT in the biscuit was 100-fold lower than the biscuit formulated with HT. Methylglyoxal, 3-DG, and glyoxal were the predominant 1,2-dicarbonyl compounds after baking but only 3-DG was significantly reduced by HT.
Asunto(s)
Alcohol Feniletílico/análogos & derivados , Extractos Vegetales/farmacología , Hojas de la Planta/química , Arginina/análogos & derivados , Arginina/análisis , Arginina/antagonistas & inhibidores , Cromatografía Liquida , Desoxiglucosa/análogos & derivados , Desoxiglucosa/análisis , Desoxiglucosa/antagonistas & inhibidores , Harina/análisis , Manipulación de Alimentos , Furaldehído/análogos & derivados , Furaldehído/análisis , Furaldehído/antagonistas & inhibidores , Ácido Gálico/farmacología , Productos Finales de Glicación Avanzada/análisis , Productos Finales de Glicación Avanzada/antagonistas & inhibidores , Lisina/análogos & derivados , Lisina/análisis , Lisina/antagonistas & inhibidores , Reacción de Maillard/efectos de los fármacos , Olea/química , Alcohol Feniletílico/farmacología , Piruvaldehído/análisis , Piruvaldehído/antagonistas & inhibidores , Quercetina/farmacología , Espectrometría de Masas en Tándem , Triticum/químicaRESUMEN
Osteoporosis is a metabolic multifaceted disorder, characterized by insufficient bone strength. It has been recently shown that advanced glycation end products (AGEs) play a role in senile osteoporosis, through bone cell impairment and altered biomechanical properties. Pentosidine (PENT), a wellcharacterized AGE, is also considered a biomarker of bone fracture. Adequate responses to various hormones, such as 1,25-dihydroxyvitamin D3, are prerequisites for optimal osteoblasts functioning. Vitamin K2 is known to enhance in vitro and in vitro vitamin D-induced bone formation. The aim of the study was to assess the effects of Vitamins D3 and K2 and PENT on in vitro osteoblast activity, to convey a possible translational clinical message. Ex vivo human osteoblasts cultured, for 3 weeks, with vitamin D3 and vitamin K2 were exposed to PENT, a well-known advanced glycoxidation end product for the last 72 hours. Experiments with PENT alone were also carried out. Gene expression of specific markers of bone osteoblast maturation [alkaline phosphatase, ALP; collagen I, COL Iα1; and osteocalcin (bone-Gla-protein) BGP] was measured, together with the receptor activator of nuclear factor kappa-B ligand/osteoproteregin (RANKL/OPG) ratio to assess bone remodeling. Expression of RAGE, a well-characterized receptor of AGEs, was also assessed. PENT+vitamins slightly inhibited ALP secretion while not affecting gene expression, indicating hampered osteoblast functional activity. PENT+vitamins up-regulated collagen gene expression, while protein secretion was unchanged. Intracellular collagen levels were partially decreased, and a significant reduction in BGP gene expression and intracellular protein concentration were both reported after PENT exposure. The RANKL/OPG ratio was increased, favouring bone reabsorption. RAGE gene expression significantly decreased. These results were confirmed by a lower mineralization rate. We provided in vitro evidence that glycoxidation might interfere with the maturation of osteoblasts, leading to morphological modifications, cellular malfunctioning, and inhibition of the calcification process. However, these processes may be all partially counterbalanced by vitamins D3 and K2. Therefore, detrimental AGE accumulation in bone might be attenuated and/or reversed by the presence or supplementation of vitamins D3 and K2.
Asunto(s)
Arginina/análogos & derivados , Colecalciferol/farmacología , Lisina/análogos & derivados , Osteoblastos/efectos de los fármacos , Vitamina K 2/farmacología , Fosfatasa Alcalina/biosíntesis , Fosfatasa Alcalina/genética , Antígenos de Neoplasias/biosíntesis , Antígenos de Neoplasias/genética , Arginina/antagonistas & inhibidores , Arginina/toxicidad , Remodelación Ósea/efectos de los fármacos , Células Cultivadas , Colágeno Tipo I/biosíntesis , Colágeno Tipo I/genética , Cadena alfa 1 del Colágeno Tipo I , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Lisina/antagonistas & inhibidores , Lisina/toxicidad , Proteínas Quinasas Activadas por Mitógenos/biosíntesis , Proteínas Quinasas Activadas por Mitógenos/genética , Osteoblastos/metabolismo , Osteocalcina/biosíntesis , Osteocalcina/genética , Osteogénesis/efectos de los fármacos , Osteoprotegerina/biosíntesis , Osteoprotegerina/genética , Ligando RANK/biosíntesis , Ligando RANK/genética , ARN Mensajero/biosíntesis , ARN Mensajero/genéticaRESUMEN
γ-Glutamyl transpeptidase (GGT, EC 2.3.2.2) that catalyzes the hydrolysis and transpeptidation of glutathione and its S-conjugates is involved in a number of physiological and pathological processes through glutathione metabolism and is an attractive pharmaceutical target. We report here the evaluation of a phosphonate-based irreversible inhibitor, 2-amino-4-{[3-(carboxymethyl)phenoxy](methoyl)phosphoryl}butanoic acid (GGsTop) and its analogues as a mechanism-based inhibitor of human GGT. GGsTop is a stable compound, but inactivated the human enzyme significantly faster than the other phosphonates, and importantly did not inhibit a glutamine amidotransferase. The structure-activity relationships, X-ray crystallography with Escherichia coli GGT, sequence alignment and site-directed mutagenesis of human GGT revealed a critical electrostatic interaction between the terminal carboxylate of GGsTop and the active-site residue Lys562 of human GGT for potent inhibition. GGsTop showed no cytotoxicity toward human fibroblasts and hepatic stellate cells up to 1mM. GGsTop serves as a non-toxic, selective and highly potent irreversible GGT inhibitor that could be used for various in vivo as well as in vitro biochemical studies.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Escherichia coli/enzimología , Lisina/antagonistas & inhibidores , Organofosfonatos/farmacología , gamma-Glutamiltransferasa/antagonistas & inhibidores , Dominio Catalítico/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Humanos , Lisina/metabolismo , Modelos Moleculares , Estructura Molecular , Organofosfonatos/síntesis química , Organofosfonatos/química , Electricidad Estática , Relación Estructura-Actividad , gamma-Glutamiltransferasa/química , gamma-Glutamiltransferasa/metabolismoRESUMEN
Histone lysine methylation can be modified by various writers and erasers. Different from other epigenetic modifications, mono-, di, and tri- methylation distinctly modulate chromatin structure and thereby contribute to the regulation of DNA-based nuclear processes such as transcription, replication and repair on their target genes depending on different sites. Modulators with opposing catalytic activities dynamically and precisely control levels of histone lysine methylation, and individual enzymes within these families have become candidate oncology targets in recent years. Until now, plenty of medicinal chemists try to pursue potent and selective inhibitor for KMTs and KDMs in order to have the potential anti-cancer agent, and several of the inhibitors have already enrolled in clinic. Here, we discuss three histone lysine methylation modulators with their inhibitors in clinical trials.
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Antineoplásicos/farmacología , Histonas/antagonistas & inhibidores , Lisina/antagonistas & inhibidores , Neoplasias/tratamiento farmacológico , Antineoplásicos/química , Histonas/metabolismo , Humanos , Lisina/metabolismo , Metilación/efectos de los fármacos , Neoplasias/metabolismoRESUMEN
AIM: The aim of this study was to examine the activation of neuronal Kv7/KCNQ channels by a novel modified Kv7 opener QO58-lysine and to test the anti-nociceptive effects of QO58-lysine on inflammatory pain in rodent models. METHODS: Assays including whole-cell patch clamp recordings, HPLC, and in vivo pain behavioral evaluations were employed. RESULTS: QO58-lysine caused instant activation of Kv7.2/7.3 currents, and increasing the dose of QO58-lysine resulted in a dose-dependent activation of Kv7.2/Kv7.3 currents with an EC50 of 1.2±0.2 µmol/L. QO58-lysine caused a leftward shift of the voltage-dependent activation of Kv7.2/Kv7.3 to a hyperpolarized potential at V1/2=-54.4±2.5 mV from V1/2=-26.0±0.6 mV. The half-life in plasma (t1/2) was derived as 2.9, 2.7, and 3.0 h for doses of 12.5, 25, and 50 mg/kg, respectively. The absolute bioavailabilities for the three doses (12.5, 25, and 50 mg/kg) of QO58-lysine (po) were determined as 13.7%, 24.3%, and 39.3%, respectively. QO58-lysine caused a concentration-dependent reduction in the licking times during phase II pain induced by the injection of formalin into the mouse hindpaw. In the Complete Freund's adjuvant (CFA)-induced inflammatory pain model in rats, oral or intraperitoneal administration of QO58-lysine resulted in a dose-dependent increase in the paw withdrawal threshold, and the anti-nociceptive effect on mechanical allodynia could be reversed by the channel-specific blocker XE991 (3 mg/kg). CONCLUSION: Taken together, our findings show that a modified QO58 compound (QO58-lysine) can specifically activate Kv7.2/7.3/M-channels. Oral or intraperitoneal administration of QO58-lysine, which has improved bioavailability and a half-life of approximately 3 h in plasma, can reverse inflammatory pain in rodent animal models.
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Canales de Potasio KCNQ/agonistas , Lisina/farmacología , Dimensión del Dolor/efectos de los fármacos , Animales , Antracenos/farmacología , Disponibilidad Biológica , Carbamatos/farmacología , Relación Dosis-Respuesta a Droga , Células HEK293 , Humanos , Lisina/antagonistas & inhibidores , Lisina/farmacocinética , Masculino , Potenciales de la Membrana/efectos de los fármacos , Fenilendiaminas/farmacología , RatasRESUMEN
BACKGROUND: In non-dialysis chronic kidney disease (CKD) patients with dyslipidemia, statin therapy is recommended to prevent cardiovascular complications. Dyslipidemia has been also shown to be an independent risk factor for the progression of CKD. However, it is still unclear whether statin therapy exerts an inhibitory effect on renal deterioration in CKD patients with dyslipidemia. The purpose of the present study was to examine possible therapeutic effects of statin add-on therapy on renal function as well as parameters of lipid and glucose metabolism, arterial stiffness and oxidative stress, in comparison to diet therapy, in CKD patients with dyslipidemia. METHODS: This study was a randomized, open-label, and parallel-group trial consisted of a 12-months treatment period in non-dialysis CKD patients with alubuminuria and dyslipidemia. Twenty eight patients were randomly assigned either to receive diet counseling alone (diet therapy group) or diet counseling plus pitavastatin (diet-plus-statin therapy group), to achieve the LDL-cholesterol (LDL-C) target of <100 mg/dl. RESULTS: The statin treatment by pitavastatin was well tolerated in all of the patients without any significant adverse events and the average dose of pitavastatin was 1.0 ± 0.0 mg daily after treatment. After the 12-months treatment period, LDL-C was significantly lower in the diet-plus-statin therapy group compared with the diet therapy group (diet vs diet-plus-statin: LDL-C, 126 ± 5 vs 83 ± 4 mg/dL, P < 0.001). On the other hand, the diet-plus-statin therapy did not significantly reduce albuminuria or delay the decline in eGFR compared with the diet therapy, and there was no relationship between the change in LDL-C and the change in eGFR or albuminuria. However, diet therapy as well as diet-plus-statin therapy exerted similar lowering effects on the pentosidine levels (diet therapy group, baseline vs 12 months: 40 ± 4 vs 24 ± 3 ng/mL, P = 0.001; diet-plus-statin therapy, 46 ± 7 vs 34 ± 6 ng/mL, P = 0.008). Furthermore, the results of multivariate regression analysis indicated that the change in pentosidine was a significant contributor to the change in eGFR (ß = -0.536, P = 0.011). CONCLUSIONS: Although statin add-on therapy did not show additive renal protective effects, the diet therapy as well as the diet-plus-statin therapy could contribute to the reduction in plasma pentosidine in CKD patients with albuminuria and dyslipidemia.
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Albuminuria/tratamiento farmacológico , Anticolesterolemiantes/uso terapéutico , Dieta/métodos , Dislipidemias/tratamiento farmacológico , Quinolinas/uso terapéutico , Insuficiencia Renal Crónica/tratamiento farmacológico , Anciano , Albuminuria/sangre , Albuminuria/dietoterapia , Albuminuria/patología , Arginina/análogos & derivados , Arginina/antagonistas & inhibidores , Arginina/sangre , HDL-Colesterol/sangre , LDL-Colesterol/antagonistas & inhibidores , LDL-Colesterol/sangre , Dislipidemias/sangre , Dislipidemias/dietoterapia , Dislipidemias/patología , Femenino , Tasa de Filtración Glomerular , Productos Finales de Glicación Avanzada/antagonistas & inhibidores , Productos Finales de Glicación Avanzada/sangre , Humanos , Lisina/análogos & derivados , Lisina/antagonistas & inhibidores , Lisina/sangre , Masculino , Persona de Mediana Edad , Estrés Oxidativo/efectos de los fármacos , Insuficiencia Renal Crónica/sangre , Insuficiencia Renal Crónica/dietoterapia , Insuficiencia Renal Crónica/patología , Triglicéridos/sangre , Rigidez Vascular/efectos de los fármacosRESUMEN
Nε-(carboxymethyl) lysine-conjugated bovine serum albumin (CML-BSA) is a major component of advanced glycation end products (AGEs). We hypothesised that AGEs reduce insulin secretion from pancreatic ß-cells by damaging mitochondrial functions and inducing mitophagy. Mitochondrial morphology and the occurrence of autophagy were examined in pancreatic islets of diabetic db/db mice and in the cultured CML-BSA-treated insulinoma cell line RIN-m5F. In addition, the effects of α-lipoic acid (ALA) on mitochondria in AGE-damaged tissues were evaluated. The diabetic db/db mouse exhibited an increase in the number of autophagosomes in damaged mitochondria and receptor for AGEs (RAGE). Treatment of db/db mice with ALA for 12 wk increased the number of mitochondria with well-organized cristae and fewer autophagosomes. Treatment of RIN-m5F cells with CML-BSA increased the level of RAGE protein and autophagosome formation, caused mitochondrial dysfunction, and decreased insulin secretion. CML-BSA also reduced mitochondrial membrane potential and ATP production, increased ROS and lipid peroxide production, and caused mitochondrial DNA deletions. Elevated fission protein dynamin-related protein 1 (Drp1) level and mitochondrial fragmentation demonstrated the unbalance of mitochondrial fusion and fission in CML-BSA-treated cells. Additionally, increased levels of Parkin and PTEN-induced putative kinase 1 protein suggest that fragmented mitochondria were associated with increased mitophagic activity, and ALA attenuated the CML-BSA-induced mitophage formation. Our study demonstrated that CML-BSA induced mitochondrial dysfunction and mitophagy in pancreatic ß-cells. The findings from this study suggest that increased concentration of AGEs may damage ß-cells and reduce insulin secretion.